Long-Term Objectives: To identify and characterize viral genes that promote directional, transneuronal spread of herpesviruses in the mammalian nervous system. This will aid the search for more delivery- specific, less pathogenic herpesvirus vectors. Specific Aims: Pseudorabies virus (PRV), a close relative to herpes simplex virus (HSV), infects and spreads within the peripheral and central nervous systems in a wide range of mammals, and is commonly used to map neuronal circuitry in the rodent brain. Previous work in our lab has identified three PRV gene products, gE, gl, and Us9, that are critical for directional spread in synaptically connected neurons; deletion of any one of these three genes blocks viral spread from pre-synaptic to post-synaptic neurons, but not vice versa. Experiments in this proposal will focus specifically on Us9, and its role in directional spread of PRV and HSV in neurons. AIM 1: To identify Us9 binding proteins that mediate axonal targeting of viral glycoproteins. AIM 2: To examine Us9 trafficking patterns within living neurons by confocal microscopy. AIM 3: To characterize the Us9 homolog of herpes simplex virus (HSV-1). Study Design: Cellular and/or viral proteins that interact with Us9 will be identified by tandem affinity purification (TAP) and subsequent mass spectroscopy analysis. PC12 cells -- a pheochromocytoma cell line that differentiates into sympathetic-like neurons in the presence of nerve growth factor - will be infected with PRV recombinant viruses that express a TAP tag fused to wild-type Us9, or a Us9 di-tyrosine mutant that does not target viral structural components to the axon of infected neurons. To examine the real-time localization of Us9 in neurons, we will fuse Us9 with photoactivatable GFP (PAGFP), and visualize its movement within primary neurons. This system will reveal several aspects of Us9 biology, specifically the kinetics/dynamics of Us9 trafficking from the Golgi to the axon, and which Us9 motifs and post-translational modifications dictate how it is targeted to the axon. Lastly, we will test whether HSV Us9 will functionally compensate for PRV Us9 in vivo using the rat eye model, as well as characterize the HSV Us9 homolog in dissociated neuronal cultures. Relevance to Public Health: Herpesvirus vectors that target specific areas of the central nervous would be efficacious for the delivery of genes that treat neurological diseases such as brain tumors (glioma), Parkinson's disease, spinal cord injury, and spinal root trauma. [unreadable] [unreadable] [unreadable]